Povidone-containing carriers for polypeptide growth factors

ABSTRACT

A liquid carrier medium is provided which is suitable for solubilizing growth factors, such as mixtures of bone morphogenetic proteins, that are found to induce an angiogenic response in ischemic tissues. The liquid medium comprises an aqueous solution of polyvinyl pyrrolidone.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 09/748,038(which is incorporated herein by reference in its entirety), which is acontinuation-in-part of U.S. Ser. No. 09/173,989, filed Oct. 16, 1998,now issued as U.S. Pat. No. 6,211,157 (Benedict et al.) and entitled“Protein Mixtures to Induce Therapeutic Angiogenesis” (which is alsoincorporated herein by reference in its entirety).

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The present invention relates to the use of polyvinyl pyrrolidone(hereinafter “povidone”) in a liquid medium for proteins andpolypeptides, especially those which function as angiogenic growthfactors.

BACKGROUND OF THE INVENTION

There are many medical circumstances in which an increase in the supplyof blood to living tissue is indicated. These include: burns and woundhealing, in which the incorporation of angiogenic factors intoartificial skin may facilitate the formation of blood vessels in thehealing wound bed and also reduce the risk of infection; cardiovasculardisease, in which repair of anginal or ischemic cardiac tissue can beeffected by the ingrowth of new blood vessels; stroke, where increasedblood supply to the brain can reduce the risk of transient ischemicattack and/or cerebral arterial deficiency; and peripheral vasculardisease, in which blood flow in the extremities is diminished. In eachcase, it is believed that the growth of new blood vessels will increasethe volume of blood circulating through the tissue in question, andcorrespondingly increase the amount of oxygen and nutrients madeavailable to that tissue.

One common cause of decreased blood flow is atherosclerosis.Atherosclerosis affects the blood vessels, including those of the heart,and is a major cause of cardiovascular disease, stroke and peripheralvascular disease. This disease may have its beginnings early in life andis initially characterized by a thickening of the arterial walls. Thisthickening typically comprises an accumulation of fat, fibrin, cellulardebris and calcium. The resultant narrowing of the lumen of theafflicted vessel is called stenosis. Stenosis impedes and reduces bloodflow. Hypertension and dysfunction of the organ or area of the body thatsuffers the reduced blood flow can result. As the buildup on the innerwall of a vessel thickens, the vessel wall loses the ability to expandand contract. Also, the vessel loses its viability and becomes weakenedand susceptible to bulging, also known as aneurysm. In the presence ofhypertension or elevated blood pressure, aneurysms will frequentlydissect and ultimately rupture.

Small vessels, such as the arteries that supply blood to the heart,legs, intestines and other areas of the body, are particularlysusceptible to atherosclerotic narrowing. When an artery in the leg orintestine is affected, the resultant loss of blood supply to the leg orsegment of the intestine may result in gangrene. Atheroscleroticnarrowing of one or more of the coronary arteries limits, and in someinstances prevents altogether, blood flow to portions of the heartmuscle. Depending upon the severity of the occlusion and its locationwithin the coronary circulation system, pain, cardiac dysfunction ordeath may result. Because the consequences of blocked arteries are soserious, reliable treatments are highly desirable.

In many instances, it is possible to correct aneurysms and stenosis ofmajor arteries using plastic reconstruction that does not require anysynthetic graft or patch materials. In other instances, such as wherethe disease is extensive and the vessel is no longer competent, theblocked or weakened portion of the vessel is usually replaced with agraft. In such case, the affected vessel section is transected andremoved and a synthetic patch, conduit or graft is sewn into its place.These types of procedures, including coronary artery bypass grafting(CABG) and percutaneous transluminal coronary angioplasty (PTCA), areroutinely performed for the purpose of alleviating ischemia.

Nevertheless, coronary artery disease alone is responsible forapproximately 550,000 deaths each year in the United States. Peripheralvascular disease results in lower limb amputation in about 150,000patients each year, with a subsequent mortality rate of 40% within twoyears of amputation. Some of the difficulty in treating arterialocclusions may lie in the fact that each of the aforementioned surgicalprocedures is associated with a certain incidence of restenosis and maynot be appropriate in certain instances. This is particularly true whenthe patient is elderly or has undergone a previous CABG or PTCAprocedure. Accordingly, in such cases a less invasive technique would bepreferred. In particular, it would be advantageous to be able tostimulate the surrounding tissue to produce for itself new vessels thatwould accommodate the volume of blood flow that has been occluded andthus compensate for the occluded vessels.

Angiogenic, or “vessel-growing” polypeptide growth factors in generalhave been the subject of much research. Such compositions offer thepromise of a non-invasive, non-surgical treatment of arterial occlusionin a variety of situations, including those noted above. However, onemajor challenge facing the development of physiological treatments basedon these materials is the lack of suitable solvents or carriers for theadministration of growth factors to living patients.

A number of solvents have been used as carriers for particularapplications within the pharmaceutical arts. Thus, for example, U.S.Pat. No. 5,981,489 (Stevenson et al.) discloses a number of non-aqueousprotic formulations of peptides. Among the solvents mentioned thereinare polyethylene glycols (PEGs), propylene glycol (PG),polyvinylpyrrolidone (PVP), methoxypropylene glycol (MPEG), glycerol,and glycofurol.

However, many solvents that adequately solubilize some pharmaceuticalpreparations and biological materials do not adequately solubilizepolypeptide growth factors. Consequently, treatments utilizing suchsolvents may not induce an angiogenic response, even if the particulargrowth factor being used is capable of inducing such a response whenproperly delivered to the body of the patient. Other solvents tend tointeract with, denature, induce crosslinking or cause other undesirablereactions in polypeptide growth factors, thus causing them to coagulateor precipitate, or otherwise rendering them pharmaceutically inactive orunsuitable. This is especially true if the solvent medium does notafford an acceptable pH range to the polypeptide growth factor (e.g., ifit is too basic). Still other potentially useful solvents do not formstable solutions with growth factors, and hence cannot be used to makeformulations having acceptable shelf stability.

Many potentially useful solvents for growth factors are alsophysiologically unsuitable. For example, investigators have used dilutesolutions of hydrochloric acid (1 to 10 mM) to solubilize certain growthfactors. See, e.g., R. J. Laham, M. Rezaee, M. Post, D. Novicki, F. W.Sallke, J. D. Pearlman, M. Simmons and D. Hung, “IntrapericardialDelivery of Fibroblast Growth Factor-2 Indices Neovascularization in aProcine Model of Chronic Myocardial Ischemia”, J. Pharmocolo. Exp. Ther.292(2), 795-802 (2000); N. Yamamoto, T. Kohmoto, W. Roethy, A. Gu, C.DeRosa, L. E. Rabbani, C. R. Smith, and D. Burkhoff, “HistologicalEvidence That Basic Fibroblast Growth Factor Enhances the AngiogenicEffects of Transmyocardial Laser Revascularization”, J. Pharmocolo. Exp.Ther. 95(1), 55-63 (2000). However, for clinical applications, acidsolvents such as dilute mineral acids are unsuitable because their usetends to cause cell damage or death in the proximity of the site ofadministration. On the other hand, saline solutions and neutral bufferedsalt solutions, which are more biologically compatible than mineralacids and which are used as solvents in some pharmaceuticalformulations, cause many growth factor proteins to precipitate or becomedenatured, thus decreasing their bioavailability or effectiveness in thetissue.

The selection of a suitable solvent for growth factors is furthercomplicated by other considerations. For example, even if a solventadequately solubilizes a growth factor and does not cause it to becomedenatured or otherwise adversely affected, the solvent may nonethelessinterfere with elution of the growth factor from the solvent medium,thereby reducing its efficacy. This may be the case, for example, if thesolvent is too viscous, or bonds to the growth factor (e.g., throughhydrogen bonding or dipole-dipole interactions) too strongly.

There is thus a need in the art for a solvent or carrier thatsolubilizes polypeptide growth factors sufficiently to render thempharmacologically useful, that does not cause them to coagulate, becomedenatured, or undergo crosslinking or other reactions that wouldadversely affect their pharmaceutical activity, that exhibits good shelfstability, that does not cause significant injury or damage to cells atthe site of administration, and that allows the growth factor toproperly elute from the solvent medium after administration to livingtissues. These and other needs are addressed by the present invention,as hereinafter described.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a composition comprisingone or more polypeptide growth factors and a carrier medium therefor.The carrier is preferably an aqueous medium which solubilizespolypeptide growth factors sufficiently to render them pharmacologicallyuseful, does not cause them to coagulate, become denatured, or undergocrosslinking or cause other reactions that would adversely affect theirpharmaceutical activity. The present invention provides mixtures,solutions and compositions that exhibit good shelf stability, do notcause significant injury or damage to cells at the site ofadministration, and allow the growth factor to properly elute from themedium upon administration to living tissues.

The carrier medium comprises at least one compound, polymer or copolymercontaining a lactam or pyrrolidone moiety, and preferably at least onecompound, polymer or copolymer containing a vinyl lactam or vinylpyrrolidone moiety. More preferably, the liquid medium comprises asolution of polyvinyl pyrrolidone, such as a polymer derived fromN-vinyl-2-pyrrolidone, in an aqueous medium. Most preferably, the liquidmedium comprises a water soluble homopolymer of N-vinyl-2-pyrrolidone,such as povidone, and water. Aqueous buffers which maintain the pH ofthe pyrrolidone moiety at or near the same pH as water may also be used,and the compositions may also include adjuvants and preservatives.

The carrier medium of the present invention may be combined with or usedas a carrier or solvent for various growth factors, proteins,polypeptides and other such biological materials, including the variousgrowth factors, matrix metalloproteinases, cytokines, and integrins thatregulate angiogenesis. Thus, for example, the liquid media of thepresent invention may be used in conjunction with Vascular EndiothelialGrowth Factor (VEGF), Transforming Growth Factor Beta (TGF-β),Fibroblast Growth Factors (FGF), Epidermal Growth Factors (EGF),angiogenin, TNF-alpha, insulin-like growth factor-1 (IGF-1),transforming growth factor α (TGF-α), platelet-derived growth factors(PDGF), Placental Growth Factors (PGF), Heparin-binding EGF-like GrowthFactors (HEGF), Hepatocyte Growth Factors (HGF), Interferon-gamma(IFN-gamma), various Bone Morphogenic Proteins (BMP),growth/differentiation factors, vascular endothelial growth factors, andmixtures of any two or more of the foregoing materials.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more detailed description of the present invention, reference willnow be made to the accompanying Figures, wherein:

FIGS. 1A-E are histological sections of blood vessels formed in thecanine myocardium following treatment with a protein mixture inaccordance with the present invention; and

FIGS. 2 and 3 are histological slides showing newly formed blood vesselsin response to the treatment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Angiogenesis is a complex process involving several different cell typesand molecular signaling events. Endothelial cells must secrete proteasesto dissolve cell-cell and cell-matrix attachments, migrate andproliferate to form new vascular branches. Although single factors suchas bFGF and VEGF have shown promise as angiogenic agents, it has beendiscovered that a more robust angiogenic response may be obtainedthrough the use of an agent that comprises a mixture of proteins, aresult which may be due in part to a synergistic effect of the combinedproteins on the subject tissue.

Such protein mixtures preferably include the angiogenic mixtures of boneproteins described in commonly assigned U.S. Ser. No. 09/748,038, inU.S. Pat. No. 6,211,157 (Benedict et al.), in U.S. Pat. No. 5,290,763and U.S. Pat. No. 5,371,191, all of which are incorporated by referenceherein in their entirety. These mixtures may vary in composition and maybe characterized in different ways. However, one way of characterizingthe preferred compositions are as mixtures of proteins having residuescomprising about 20-25 mole percent of acidic amino acids [ASP(+ASN) andGLU(+GLN)]; about 10-15 mole percent of hydroxy amino acids (SER andTHR); about 35-45 mole percent aliphatic amino acids (ALA, GLY, PRO,MET, VAL, ILE, and LEU); about 4-10 mole percent aromatic amino acids(TYR and PHE); and about 10-20 mole percent basic amino acids (HIS, ARGand LYS). More preferably, the angiogenic protein mixture aminopreferably has an amino acid composition of about 23.4 mole percent ofacidic amino acids [ASP(+ASN) and GLU(+GLN)]; about 13.5 mole percent ofhydroxy amino acids (SER and THR); about 40.0 mole percent aliphaticamino acids (ALA, GLY, PRO, MET, VAL, ILE, and LEU); about 6.8 molepercent aromatic amino acids (TYR and PHE); and about 16.6 mole percentbasic amino acids (HIS, ARG and LYS) (TRP, CYS and ½ CYS were notmeasured and are not included in the calculation of mole percent).

Another way of characterizing the preferred protein mixtures are asmixtures containing at least two, and preferably all, of the followingpolypeptide growth factors: TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-3, BMP-4,BMP-5, BMP-6, BMP-7, and FGF-1. Preferred mixtures may also contain boneand/or serum proteins such as osteocalcin, osteonectin, albumin,transferrin, and APO-A1-LP, histones, RL-32, RS2D, and SPP24. Theseangiogenic growth factor mixtures may be employed to achieve a naturalbypass effect by injecting the mixture into tissue in need of increasedblood flow. Thus, for example, the mixtures may be used to induce vesselgrowth so as to heal a heart artery that has been blocked, or to promoteangiogenesis in ischemic tissue so as to assist in recovery.

The bone-derived angiogenic protein (BDAP) mixtures preferred for use inthe present invention are preferably administered to ischemic tissue ina suitable carrier or medium. These mixtures may be applied directly,e.g., through injection into the ischemic tissues, or indirectly, e.g.,through intravenous administration. In some instances, it may be desiredto apply the angiogenic factor in a carrier or medium that allows it tobe absorbed quickly, while in other instances it may be desired to applythe angiogenic factor in a controlled, time-release manner. In stillother instances, a single dose or other variation may be preferred. Ingeneral, the preferred carrier or medium will vary depending on thedesired clinical application and/or site of administration.

Various materials may be combined with growth factors, proteins,polypeptides and other biochemical substances in mixtures and treatmentsmade in accordance with the present invention. These materials may actto solubilize, stabilize, increase the bioavailability of, renderpharmacologically useful, and/or facilitate the administration of suchsubstances to a living subject. Such materials include, for example,polyactic acid, polyglycolic acid and its copolymers, collagen,PLURONIC® (polyoxyalkylene ether copolymer surfactant), vinylamidepolymers and copolymers, vinylimide polymers and copolymers, andcompounds, polymers and copolymers containing lactam or pyrrolidonemoieties.

Typically, materials used in liquid media in accordance with the presentinvention will be materials that solubilize (or aid in thesolubilization of) desired proteins, polypeptides, growth factors, ormixtures of the foregoing to a degree sufficient to render thempharmacologically useful and/or to increase their bioavailability. It isalso preferred that materials employed as liquid media do not causesignificant injury or damage to cells at the site of administration, andallow the material being solubilized to properly elute from solutionupon administration to living tissues.

Liquid media comprising vinylamide polymers and copolymers, vinylimidepolymers and copolymers, vinyl lactam monomers or copolymers, andcompounds, polymers and copolymers containing lactam or pyrrolidonemoieties may be used in various applications of the present invention.Preferably, however, the liquid media used in the present inventioncomprise at least one polymer derived from a vinyl pyrrolidone monomeror having a pyrrolidone moiety in its molecular structure. Morepreferably, these media comprise a polymer derived fromN-vinyl-2-pyrrolidone. Most preferably, these media comprise a watersoluble homopolymer of N-vinyl-2-pyrrolidone such as povidone.

The use of povidone (polyvinylpyrrolidone) in media compositions made inaccordance with the present invention is especially preferred since itis commercially available in various pharmaceutical grades havingvarious molecular weights, has good solubility in both aqueous andvarious organic media, and is found to solubilize a variety of proteins,polypeptides, growth factors, and mixtures of the foregoing sufficientlywell to render them pharmacologically useful and/or to increase theirbioavailability. Moreover, with proper choice of parameters such asviscosity, it is possible to formulate liquid media comprising povidonewhich allow the proper elution of growth factors from the media uponapplication of the same to living tissues. In some applications,parameters such as the viscosity of the liquid medium may be manipulatedto advantageously control or achieve a desired rate of elution of activeingredients from the liquid medium.

Pharmaceutical grades of povidone suitable for use in the presentinvention are available commercially from International SpecialtyProducts, Inc., Wayne, N.J., under the trademark Plasdone®. Propertiesof commercially available povidone are known in the art and may beobtained from a number of references, including “PVP: A Critical, Reviewof the Kinetics and Toxicology of Polyvinylpyrrolidone (Povidone),”Robinson, Sullivan, Borzelleca, and Schwartz, Lewis Publishers, Inc.,121 S. Main Street, P.O. Drawer 519, Chelsea, Mich., and “PVPpolyvinylpyrrol idone Polymers”, GAF(ISP) Technical Bulletin, 2302-203SM-1290. Each of these grades may be employed in the liquid media of thepresent invention.

Povidone is available in a wide range of molecular weights andviscosities, reflecting polymers that consist of varying numbers ofmonomeric units. The different molecular weight grades of povidone arefrequently characterized by their K-value, which is a measure of themean molar mass of a polymeric substance and is derived from therelative viscosities of polymer solutions. The K-value of a polymer isrelated to its molecular weight by the Fikentscher equation (EQUATION1), which provides that: $\begin{matrix}{{\log\frac{\eta_{c}}{\eta_{0}}} = {\left( {\frac{75\quad k^{2}}{1 + {1.5\quad{kc}}} + k} \right)c}} & {{EQUATION}\quad 1}\end{matrix}$where c is the concentration (in g/100 ml) of the polymer in solution,TV is the viscosity of the solution, η₀ is the viscosity of the solvent,and K=1000 k, wherein K is the K-value of the polymer. The Fikentscherequation is described in greater detail in H. Fikentscher,Cellusochemie, 1932, 13, 58-64 and 71-74.

K-values for a polymeric material can be obtained by dissolving thematerial in an appropriate amount of solvent so as to produce a dilutesolution (e.g., about 1% by weight of the polymeric material, based onthe total weight of the solution). The viscosity of the solution is thendetermined at 25° C., using, for example, a Höppler falling-ballviscometer or other means known in the art. The viscosity of the puresolvent is determined using the same viscometer. These values are theninserted into EQUATION 1, which is solved for k (and therefore K).

TABLE II describes the physical characteristics, including K-values, ofsome commonly available grades of povidone that may be used in thepresent invention: TABLE II Commercial Grades of Povidone TypicalTypical Molecular Viscosity² Product K-Value Weight¹ (cP) Plasdone ®C-15 16-18 (16.1, 16.3) 8,000 1.5 Plasdone ® C-30 29-32 58,000 2.5Plasdone ® K-25 24-26 34,000 2.0 Plasdone ® K-29/32 29-32 58,000 2.5Plasdone ® K-90/D 85-95 (90) 1,300,000 55.0¹Weight average, as determined by light scattering.²5% solution in de-ionized water. Measured on a Brookfield LVTviscometer, 60 rpm @ 25° C.The range of K-values noted in TABLE II reflects the productspecifications set by the manufacturer (International SpecialtyProducts, Inc., Wayne, N.J.), it being understood that the K-values ofthese products will vary from one product batch to another within thespecified ranges. The parenthetical values are the actual K-valuesdetermined for particular product samples used in some of theexperiments described herein, infra, as specified on the certificates ofanalysis that accompanied the samples. While povidone of varyingK-values may be used in the practice of the present invention, theK-value of the povidone is preferably within the range of about 12 toabout 100, more preferably within the range of about 15 to about 35, andmost preferably within the range of about 16 to about 18.

Pyrrolidone-containing polymers useful in the present invention may havea variety of molecular structures. Thus, for example, they may be cappedon one or more ends with at least one pyrrolidone moiety, they may haveat least one pendant pyrrolidone moiety, or they may have a repeatingunit or sequence comprising at least one pyrrolidone moiety. The polymermay also be a random, graft or block polymer or copolymer based on atleast one vinyl pyrrolidone monomer, comonomer or copolymer. Thepolymers used in liquid media in the present invention which are derivedfrom vinyl pyrrolidone may be grafted with lipophilic or hydrophilicradicals to make them more lipophilic or hydrophilic, respectively.

Various copolymers of vinyl pyrrolidone may also be employed in thepractice of the present invention. These include, for example,copolymers formed from comonomers such as vinylacetate, ethyl acrylate,methyl acrylate, methyl methacrylate, dimethylaminoethyl methacrylate,acrylamide, methacrylamide, acrylonitrile, ethylene, styrene, maleicanhydride, acrylic acid, sodium vinylsulfonate, vinyl chloride,vinylpyridine, trimethyl (siloxy) vinylsilane, vinyl propionate, vinylcaprolactam, and methyl vinyl ketone. Such copolymers may be synthesizedthrough polymerization in a solution, emulsion or suspension with a freeradical initiator such as a peroxy or azo compound, through the use oftransition metal catalysts, by photopolymerization techniques, or byother means as are known to the art.

Various monomers, polymers, and copolymers which are useful in thepractice of the present invention or in the synthesis of materialsuseful in the present invention may be derived from vinyl pyrrolidonemonomers in which the pyrrolidone ring and the vinyl functionality maybe coupled to each other either directly or through various intermediatestructures or moieties. Thus, for example, they may be joined by way of(a) a covalent bond; (b) an alkylene linkage, such as, for example, amethylene, ethylene, or propylene linkage; (c) an arylene linkage; (d)an alkylarylene linkage; (e) one or more hetero atoms, such as, forexample, oxygen, nitrogen, or sulfur; (f) a functional group, such as,for example, a carboxyl, carbonyl, or amino linkage; or (h) acombination of any two or more of the foregoing. Preferably; thepyrrolidone ring and the vinyl functionality are joined together by wayof a covalent bond between one of the vinyl carbon atoms and thenitrogen atom of the pyrrolidone ring. The vinyl moiety and pyrrolidonering may also have various substituents. Such substituents may include,for example, alkyl groups such as, for example, methyl groups, orfunctional groups such as, for example, hydroxy groups. Thesesubstituents may also include deuterium for use in applications where itmay be desirable to trace the progression or bioaccumulation, if any, ofthese materials in the body of a subject.

The vinyl pyrrolidone polymers and other polymeric materials useful inliquid media in accordance with the present invention may have variousmolecular weights. The properties of these polymers are found to varywith average molecular weight. Accordingly, the preferred range ofaverage molecular weights will vary from one application to another, andwill depend in part on the particular growth factor(s) or othertreatments, substances or materials which need to be solubilized.However, in most applications, the average molecular weight will be fromabout 1 kD to about 130 kD and the material will comprise between 12 and1350 monomeric units. Preferably, the molecular weight will be fromabout 1 kD to about 100 kD, more preferably from about 2.5 kD to about90 kD, and most preferably from about 2.5 kD to about 20 kD.Compositions having a molecular weight of less than about 20 kD areespecially preferred because they are readily cleared from the body,while some higher molecular weight compositions. (especially in the caseof povidone) are found to undergo bioaccumulation.

The viscosity of the liquid medium will typically be less than about 3centipoise (cP), preferably less than about 2.5 cP, more preferably lessthan about 2.0 cP, and most preferably less than about 1.5 cP (measuredat 60 rpm@ 25° C.), as viscosities within these ranges are typicallyfound to facilitate proper elution of growth factors and other suchmaterials from the solvent medium and into the tissues of a treatedsubject. Moreover, viscosities within these ranges allow application ofgrowth factors to a living subject through intravenous routes and byother non-invasive procedures. However, it is to be understood thathigher viscosities may be warranted or desirable in certain situations,as, for example, where it would be desirable to apply growth factors andother such materials as a topical gel.

The compositions made in accordance with the present invention willtypically have a pH within the range of about 1 to about 6, preferablywithin the range of about 4 to about 6, and most preferably of about4.5. In the preferred pH range, the liquid medium will adequatelysolubilize many of the generally acidic growth factors withoutdenaturing them or significantly affecting their activity in vivo, andthe pH of the composition will not be so low as to cause necrosis ordamage to living tissues, especially at the site of administration.

When vinyl pyrrolidone polymers are used in compositions of the presentinvention, they may in some instances be used in conjunction with otherpolymers, materials, solvents, or cosolvents. Thus, for example, thesepolymers may be used to form blends, mixtures, complexes, suspensions orpolymeric networks with other polymers or materials.

As previously noted, the vinyl pyrrolidone polymers and other materialsdescribed above as being useful in the present invention may be used inliquid media for various growth factors, proteins, polypeptides andother such biological materials. Such biological materials include, forexample, the novel mixtures of growth factors as have been describedherein, as well as the various growth factors, matrixmetalloproteinases, cytokines, and integrins that regulate angiogenesis.The latter group includes Vascular Endiothelial Growth Factors (VEGF),Transforming Growth Factors (TGF-beta), Fibroblast Growth Factors (FGF),Epidermal Growth Factors (EGF), angiogenin, and TNF-alpha. Other growthfactors with which these materials may be used include Placental GrowthFactors (PGF), Heparin-binding EGF-like Growth Factors (HEGF), GrowthDifferentiation Factors (GDF), Hepatocyte Growth Factors (HGF), andInterferon-gamma (IFN-gamma).

The vinyl pyrrolidone polymers and other materials described above mayalso be used in accordance with the present invention in liquid mediafor various other biological materials, including various BoneMorphogenic Proteins-(BMP) such as BMP-1 and the TGF-beta superfamily ofproteins, including BMP-2-alpha, BMP-2-beta, BMP-3, BMP-3b, BMP-4,BMP-5, BMP-6, BMP-7, BMP-8, BMP-8b, BMP-9, BMP-10, BMP-11, BMP-12,BMP-13, BMP-14, BMP-15, TGF-beta-1, TGF-beta-2, TGF-beta-3, TGF-beta-4,and TGF-beta-5; growth/differentiation factors such as GDF-1, GDF-3,GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-9B, GDF-10, GDF-11, and GDF-15;vascular endothelial growth factors such as VEGF-121, VEGF;-165,VEGF-183, VEGF-189, and VEGF-206; mitogenic proteins such as MP-52 (alsocalled GDF-5) and MP-121; and mixtures of any two or more of theforegoing materials. Many of these materials have been at leastpartially characterized in the art, and some instances the completenucleotide, DNA and/or amino acid sequences of these materials has beenderived. Thus, for example, information regarding the nucleotide, DNAand amino acid sequences of MP-52 and MP-121 can be found in U.S. Pat.No. 6,120,760 (Hötten et al.), U.S. Pat. No. 6,197,550 (Hötten et al.),and U.S. Pat. No. 5,994,094 (Hötten et al.).

When liquid media comprising the vinyl pyrrolidone polymers and othermaterials described above are used in conjunction with various growthfactors, proteins, polypeptides and other such biological materials ormixtures of biological materials as described above, the vinylpyrrolidone polymers and other such materials may be present in themedia at various concentrations. Typically, however, the vinylpyrrolidone polymers and other such materials will be provided in wateror an aqueous buffer capable of maintaining the pH within desired rangesthat completely solubilize the polypeptide growth factors. In addition,the vinyl pyrrolidone polymers are provided in the water or aqueousbuffer at concentrations ranging from the minimum concentration requiredto properly solubilize the desired growth factor or other biologicalmaterial so as to produce a pharmacologically effective solution, up tothe maximum solubility of the vinyl pyrrolidone polymer or other suchmaterial in the liquid medium.

Persons of skill in the art will appreciate that the optimalconcentration of the vinyl pyrrolidone polymers and other such materialsmay vary, and may depend on factors such as the condition of the subjectto which the solution is to be administered, the physiological effectwhich is desired, the desired viscosity of the treatment solution, andregulatory restrictions. In most applications, however, theconcentration of these vinyl pyrrolidone polymers and other suchmaterials will be from about 0.1% weight/volume to about 70%weight/volume. Preferably, the concentration of these materials will befrom about 0.1% weight/volume to about 50% weight/volume; morepreferably, the concentration of these materials will be from about 0.1%weight/volume to about 25% weight/volume; even more preferably, theconcentration of these materials will be from about 0.1% weight/volumeto about 15% weight/volume; even more preferably, the concentration ofthese materials will be from about 0.5% weight/volume to about 2.5%weight/volume; most preferably, the concentration of these materialswill be about 1.0%. In the following examples, unless stated otherwise,all concentrations of povidone are in g/ml. As used herein, %weight/volume refers to the number of grams of the designated materialdissolved in sufficient liquid (usually water) to yield 100 ml ofsolution. Thus, a 1% aqueous povidone (polyvinylpyrrolidone) solutionconsists of 1 g of povidone dissolved in sufficient water (or aqueousbuffer) to yield 100 ml of solution.

The concentration and average molecular weight of the vinyl pyrrolidonepolymers and other such materials described herein will typically bechosen to provide a medium that is sufficiently acidic to solubilize thegrowth factors and other biochemical materials employed in the mixturesand treatments described herein, while not being so viscous as to hinderthe effective administration of these materials by the desired route orto prevent sufficient elution of these materials from the media asrequired for effective treatment.

Preferably, the vinyl pyrrolidone polymers and other such materialsdescribed above for use in liquid media in accordance with the presentinvention will be present in these media as mixtures with water or anaqueous buffer capable of maintaining a desired (typically acidic) pHrange. Hence, the maximum amount of the vinyl pyrrolidone polymer orother such material present in the liquid medium will frequently bedictated at least in part by the solubility of the materials in water.The liquid medium may also contain other materials, including, but notlimited to, excipients, surfactants (including, for example, non-ionicsurfactants such as Tween 20) preservatives (including, for example,antimicrobial agent such as methyl or propyl parabens or benzylalcohols), and cosolvents.

The methods and compositions disclosed and claimed herein can be madeand executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to the methodand in the steps or in the sequence of steps of the method describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentswhich are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

The following examples are intended to be merely illustrative, and donot limit the scope of the claimed invention.

EXAMPLE 1 Canine Myocardial Angiogenesis Pilot Study

This example illustrates the efficacy of BDAP solubilized in a 70%aqueous povidone (K-90) solution in treating ischemic myocardialtissues.

Four adult mongrel dogs of either sex, weighing 21-26 kg, wereanesthetized and a left thoracotomy performed through the fifthintercostal space. All visible epicardial collaterals connecting LADartery diagonals to circumflex or right coronary arteries were ligatedto minimize collateral flow to the LAD artery territory and an ameroidconstrictor was placed on the proximal to the first diagonal branch.After completing the procedure, BDAP was applied to both the ischemicand non-ischemic left circumflex (LCX) coronary artery region in threedifferent concentrations and using three different methods ofapplication, for a total of nine injections in each region for eachanimal. Thus, 0, 10 or 100 μg BDAP was injected in 1) a 0.1 cc volume of70% ISP Plasdone® povidone having a K value of about K-90, 2)encapsulated in polymer microspheres suspended in povidone, or 3)dissolved/suspended in collagen gel for a total of nine injections. Thechest was closed and the animal was allowed to recover.

In order to provide an index of cellular proliferation at multiple timepoints after the initial surgery, bromodeoxyuridine (BrdU, 25 mg/kg,Sigma, St. Louis, Mo.) was administered subcutaneously on post-operativedays 2, 4, 6, 8, 10, 12, 14 and 21. After two or six weeks, the dogswere euthanized and the hearts explanted and cut into samples. Sampleswere fixed and serial sections, 4-5 microns thick, were cut and stainedwith Masson's trichrome stain to evaluate the general morphology of themyocardium. Sister sections were stained using standardimmunohistochemical techniques with antibodies against bromodeoxyuridine(BrdU), PC10 proliferating cell nuclear antigen (PCNA), alpha smoothmuscle actin (SMA) and Factor VIII using standard techniques.

Initial histological data (FIGS. 1A-E) indicate that 10 or 100micrograms of BDAP suspended in 0.1 cc 70% ISP Plasdoneo K-90 povidonestimulated blood vessel formation within two weeks post injection.Whereas control sections showed no significant vessel formation and theneedle track was visible, BDAP-treated sections had several newly formedblood vessels, as evidenced by Masson's trichrome staining (FIG. 1A).Immunohistochemical staining demonstrated that these vessels are linedwith endothelial cells (dark stain FIG. 1B) and surrounded by a layer ofsmooth muscle cells (brown stain FIG. 1C). Furthermore, PCNA- andBrdU-stained sections (FIGS. 1D-E) indicated that these vascularendothelial and smooth muscle cells were actively proliferating. Thus,based on the qualitative results of the canine study, it was concludedthat BDAP stimulates formation of new differentiated blood vesselsapproximately 50-100 μm in diameter in canine myocardium. The collagencarrier appeared to induce cartilage formation at the site of theinjection. The high molecular weight povidone used in these studiesappeared to be accumulating in the tissue at the site of the injectionand was not metabolized at sacrifice.

EXAMPLE 2 Large Scale Canine Myocardial Ischemia Study

This example illustrates the efficacy of various concentrations of BDAPin a 1% aqueous C-15 povidone solution in treating ischemic myocardialtissues.

The purpose of this study was to determine the effects ofintramyocardial injections of Sulzer's Growth Factor mixture (BDAP, alsoknown as ProVasc™) in a canine model of chronic myocardial ischemia.Thirty-eight (38) dogs underwent ameroid constrictor placement on theproximal LAD and ligation of visible epicardial vessels collateralizingthe LAD territory. Three weeks later, during a second surgery, animalshad intramyocardial injections of a 1% povidone (ISP Plasdone® C-15)solution containing either placebo, BDAP at a concentration of 1 μg/ml,or BDAP at a concentration of 10 μg/ml. Each injection consisted of 0.15ml of treatment solution, and injections were made at a spatial densityof ˜1/cm² over the LAD region. Group assignments were random andinvestigators were blinded to group assignment until after the analysisof all test results. Each animal tested survived for an additional 6weeks. Assessments of regional blood flow (by colored microspheres),angiography and echocardiography (rest and stress) were performed priorto and after treatment. Histology and necropsy were performed aftersacrifice.

Results of this prospective, blinded, multifaceted assessment of theeffects of BDAP solubilized in a 1% C-15 povidone solution showed thatthe treatment solution has a significant effect on vascular growthassessed histologically and by angiographic criteria. There was nosignificant effect on blood flow during maximal vasodilatory stress,though technical limitations resulted in inclusion of only a smallnumber of studies for the analysis of maximal blood flow. There was aslight reduction in regional wall motion score during maximum dobutaminestress in the high concentration group, though global resting functionwas not influenced by treatment.

EXAMPLE 3 Porcine Myocardial Ischemia Study

This example demonstrates the effectiveness of a solution of BDAP in 5%aqueous C-15 povidone at treating ischemic tissues using a porcinemodel.

Twelve pigs (mean body weight 73±4.6kg) were included in the study. Theanimals were premedicated with ketaminol (10 mg/kg) and atropine (2mg/kg) injected intramuscularly. A vascular access was establishedthrough a vein in the ear. The animals were anaesthetized with sodiumthiopental (5 m/kg) through this venous line and were intubated, andanaesthesia was maintained by intravenous administration of thiopentalas needed. The animals were ventilated with room air. A left lateralthorocotomy was performed through the fifth intercostal space. Thepericardium was opened and reflected to form a cradle for suspending theheart. In all animals, five triads of laser channels were drilled at themid-height of the left lateral wall. Within each triad, the channelswere 1 cm apart. The external opening of each channel was marked with anon-resorbable stitch in order to identify them later for histology. Theanimals were allocated randomly to receive an injection of either of 100micrograms of growth factor in 0.1 ml of 1% povidone (ISD Plasdone®C-15) within the triads, or of the same amount of the carrier alone. Atthe end of the operation, the thoracotomy was closed on a chest tube,which was removed after weaning the animal from the ventilator. After 1month, the animals were sacrificed and the hearts rapidly excised forhistology.

As shown in FIG. 3, immunostaining for Factor VIII in sections of theischemic myocardium taken from the animals treated with TMR and BDAP in5% C-15 povidone indicated an increase in the number of vascularendothelial cells compared to no treatment or treatment with TMR alone.Thus, BDAP in 5% C-15 Plasdone stimulated the formation of new bloodvessels in the subject animals.

EXAMPLE 4 Canine Myocardial Ischemia Study

This example demonstrates the effectiveness of a solution of GDF-5 in 1%aqueous C-15 povidone at treating ischemic tissues.

The experiment of EXAMPLE 1 was repeated using a solution of GDF-5solubilized in 1% povidone (ISD Plasdone® C-15) as the treating solutionin each case. As in EXAMPLE 1, BrdU-stained sections of the smoothmuscle cells and BrdU positive cells in sections of the ischemicmyocardium taken from the animals indicated that these vascularendothelial and smooth muscle cells were actively proliferating, thusindicating that GDF-5 in 1% C-15 povidone stimulated the formation ofnew differentiated blood vessels in the subject (FIG. 2).

The above-noted experimental results are summarized in TABLE 3. As theresults demonstrate, aqueous solutions of polyvinyl pyrrolidone ofvarying molecular weight and at various concentrations can be used tosolubilize various growth factors at various dosage levels. Theresulting solutions have been shown to induce an angiogenic response inporcine and canine subjects, and hence would be expected to induce asimilar response in human subjects. TABLE 3 Experimental Summary GrowthFactor Povidone Concen- Concen- Povidone EX- Growth tration trationMolecular AMPLE Factor(s) (mg/cc) (% wt/vol) Weight Subject 1 BDAP 0,0.1, 70%  K-90 Canine 1.0 (≈1,300,000 2 BDAP 0, 1, 10 1% C-15 Canine(≈8,000) 3 TMR-BDAP 1.0 5% C-15 Porcine 4 GDF-5 0, 0.5, 1% C-15 Canine2.5, 5.0

Although various embodiments of the present invention are specificallyillustrated and described herein, it will be appreciated thatmodifications and variations of the invention are covered by the aboveteachings and are within the purview of the appended claims withoutdeparting from the spirit and intended scope of the invention. Forexample, while it is preferred that the vinyl pyrrolidone polymers andother such materials described herein be incorporated into a liquidmedium, persons skilled in the art will appreciate that, in someinstances, these materials could also be advantageously employed inother media, such as, for example, solids, gels, or liquid crystals.Furthermore, the, examples set forth herein should not be interpreted tolimit the modifications and variations of the invention covered by theclaims, but are merely illustrative of some such possible variations.

1-31. (canceled)
 32. A growth factor composition, comprising: apolypeptide of the TGF-β superfamily, and a carrier comprising a vinylpyrrolidone polymer having a molecular weight of from about 2.5 kD toabout 20 kD, and a solvent selected from the group consisting of waterand aqueous buffer solutions, wherein the weight ratio of thepolypeptide to the vinyl pyrrolidone polymer is 1:10 or less and thecomposition is capable of promoting angiogenesis when administered to aliving subject at a site in need of such angiogenesis.
 33. Thecomposition of claim 32, wherein the composition is a liquid having aviscosity of less than about 3 cP.
 34. The composition of claim 33,wherein the composition is a liquid having a viscosity of less thanabout 2.5 cP.
 35. A method of promoting soft tissue regeneration in aliving subject, comprising: administering to the soft tissue a growthfactor composition comprising a polypeptide of the TGF-β superfamily anda carrier comprising a vinyl pyrrolidone polymer having a molecularweight of from about 2.5 kD to about 20 kD and a solvent selected fromthe group consisting of water and aqueous buffer solutions, wherein theweight ratio of the polypeptide to the vinyl pyrrolidone polymer is 1:10or less and angiogenesis is induced in the soft tissue.
 36. The methodof claim 35, wherein the soft tissue comprises myocardial tissue. 37.The method of claim 36, wherein administering comprises injecting thecomposition into the myocardial tissue.
 38. The method of claim 37,wherein the composition is a liquid having a viscosity of less thanabout 3 cP.
 39. The method of claim 38, wherein the composition is aliquid having a viscosity of less than about 2.5 cP.
 40. The method ofclaim 39, wherein the composition is a liquid having a viscosity of lessthan about 2 cP.
 41. The method of claim 40, wherein the composition isa liquid having a viscosity of less than about 1.5 cP.
 42. The method ofclaim 35, wherein the living subject is human.
 43. The method of claim42, wherein administering comprises injecting the growth factorcomposition into the living subject's body.
 44. The method of claim 42,wherein administering comprises injecting the growth factor compositioninto the living subject's heart.
 45. The method of claim 42, whereinadministering comprises subcutaneous administration.
 46. The method ofclaim 42, wherein administering comprises intramuscular administration.47. The method of claim 42, wherein administering comprises intravenousadministration.
 48. A method for treating ischemic tissue, comprisingthe step of administering to a patient in need thereof, a growth factorcomposition comprising: a polypeptide of the TGF-β superfamily, and acarrier comprising a vinyl pyrrolidone polymer having a molecular weightof from about 2.5 kD to about 20 kD, and a solvent selected from thegroup consisting of water and aqueous buffer solutions, wherein theweight ratio of the polypeptide to the vinyl pyrrolidone polymer is 1:10or less and the composition is capable of promoting angiogenesis whenadministered to a living subject at a site in need of such angiogenesis.49. The method of claim 48, wherein the ischemic tissue comprisesmyocardial tissue.
 50. The method of claim 48, wherein administeringcomprises injecting the growth factor composition into the ischemictissue.
 51. The method of claim 48, wherein the growth factorcomposition is a liquid having a viscosity of less than about 3 cP.